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Bac e iophage I ola ion

Bacteriophage Isolation
A e di c ed i c a , bacteriophage a e i e ha i fec bac e ia (bac e i hage ea bac e ia ea e ). Bac e i hage ha i fec E. c li e i e a e efe ed coliphage. Ge e a , bac e i hage a e efe ed i a phage. A i e f a i e , hage ca e ica e i hi h ce . I he d , c i hage ca e ica e i hi E. c li. Phage a ach a ece he face f a bac e ia ce i de i i ia e a i fec i . Thi i e ac i be ee he hage a d ece i e ecific - a gi e hage e i bi d a specific receptor molecule. Th , a hage a e a i e. A e ha e ee i he ab., a E. c li a e a i e. Va iabii e i i he g h c e a d a ibi ic e i a ce f diffe e E. c li i a e . O e c d h he i e ha he e i a e f E. c li a e e diffe e ce i hei face ec e a d, a a e , diffe e ce i he hage ece ee hei face ( e e be - he e face ec e e e ei a f c i f he bac e ia; he d e i he ce face e e a a fe f hage!). T e a i e if, i fac , ch ece diffe e ce d e i , e i c d c a hage i g e e i e . Each e i i a e a hage ha g eadi i he /hi E. c li i a e. The , e i i e iga e he he each f he e hage i a e i ca ab e f e ica i g i hi he he E. c li i a e . A he e d f hi e e i e , e i be ab e d ce a ab e ha h he e i i i e i a ce f each e ' E. c li each f he hage i a e . Each g a i e c aged de e a d c d c addi i a e e i e i h hei hage a d E. c li i a e i a a e ea e ab he di e i f he e i a e . F f he e e e i e , g ca i' . Each e , h e e , ha e a e iched hage e a a i b e ee (A i 2 3).

Bacteriophage Isolation
Each e i i a e a bac e i hage ecific f hi he i a e f E. c li. I a e he hage f ig he di ec i ided. Beca e f he e a e e h a d e i e 1 2 da i c ba i be ee he , e i d he ced e ge he a a c a . Ra he , e i g e he c da . Y ca ca he hage i a i acc di g ched e. The e i e e i ha complete Da 4 in time for lab on April 2 or 3. Obtaining bacteriophage N i i g , bac e i hage ca be f d i ace he e e h e i . Beca e c c li a a h ga i , he e hage ca be f d i ace he e E. c li a e e if . We a e e e ha a f ha b e E. c li (a d he bac e ia) i e i e i e . Ob c li ea e b die . Th , e age c ai E. c li f e e ei . A d, a a e , c ai e c i hage. I ha e b ai ed a e age a e f a e ia , e i i a e diffe e ai he R c Ri e f hage. a e ea e a i M e i e. F

i hage e E. ea ie i he i , h e E. e age a hi

Isolation of bacteriophage Da 1: I c a e 5 L ic b h ih E. c li. A e did bef e, a d a fe i he be f b h. I c ba e e igh a 37 C.

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Da 2: Inoc la e ano he be i h 4.5mL e age, 0.5mL of o o e nigh E. c li c l e, and 0.5mL of 10 p ic o b o h ( ha do e mean b '10 p ic o b o h'? Wh do e e 10 TSB fo hi ep?). o C. D ing hi inc ba ion, phage in he e age ample capable of binding o o Inc ba e 24-48 ho a 37 E. c li ill eplica e in and e en all l e he bac e ia. Th , hi ep e e a a mean of amplif ing he phage ha can infec o bac e ia. Cen if ge o be in he able op cen if ge fo 10 min e a 2500 pm. Re e be ba a ce he ce if ge! Thi cen if ga ion ep ho ld ca e he bac e ia and o he cell deb i o fo m a pelle a he bo om of he be. Take p 3mL of he pe na en in o a 5 cc inge. A ach a 0.22 mic on fil e o he ba e of he inge and fil e he pe na en in o a e ile be. Thi fil e ill p e en an emaining bac e ia f om pa ing h o gh, b ill allo phage o pa h o gh. Label he fil a e "En iched phage p ep." S o e in he ef ige a o . Se p ano he o e nigh c l e of o E. c li.

Da 3: T an fe 100 mic oli e of an o e nigh E. c li g o h on o he cen e of a 60 mm Pe i pla e con aining p ic o aga ( he e pla e ill be in he ef ige a o ). Wi h a ben gla od, p ead he bac e ia o e he pla e. Ne , place 10 mic oli e of o en iched phage p ep in he cen e of he pla e. Inc ba e a 37o C o e nigh . Da 4: E amine o pla e. Beca e e added ho and (o ma be million !) of bac e ia o he pla e, e ill no ee i ola ed colonie . Ra he a confl en la n of bac e ia ill co e he aga . The phage, ho gh, ill infec and kill he bac e ia in he cen e of he pla e, c ea ing a i ible egion of no bac e ia - a a e. Thi imple e pe imen demon a e ha o ha e i ola ed phage capable of infec ing o ain of E. c li. Re n he pla e o he inc ba o and inc ba e i fo e e al mo e da . Do o no ice an hing n al? O he da bef e e ee ' ab, i c a e a be f E. c li i a e a d i c ba e e igh a 37 C. ic b h (a i ae 5 L) i h

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